A Proteomic Investigation of Hepatic Resistance to Ascaris in a Murine Model

The helminth Ascaris causes ascariasis in both humans and pigs. Humans, especially children, experience significant morbidity including respiratory complications, growth deficits and intestinal obstruction. Given that 800 million people worldwide are infected by Ascaris, this represents a significant global public health concern. The severity of the symptoms and associated morbidity are related to the parasite burden and not all hosts are infected equally. While the pathology of the disease has been extensively examined, our understanding of the molecular mechanisms underlying resistance and susceptibility to this nematode infection is poor. In order to investigate host differences associated with heavy and light parasite burden, an experimental murine model was developed utilising Ascaris-susceptible and -resistant mice strains, C57BL/6J and CBA/Ca, respectively, which experience differential burdens of migratory Ascaris larvae in the host lungs. Previous studies identified the liver as the site where this difference in susceptibility occurs. Using a label free quantitative proteomic approach, we analysed the hepatic proteomes of day four post infection C57BL/6J and CBA/Ca mice with and without Ascaris infection to identify proteins changes potentially linked to both resistance and susceptibility amongst the two strains, respectively. Over 3000 proteins were identified in total and clear intrinsic differences were elucidated between the two strains. These included a higher abundance of mitochondrial proteins, particularly those associated with the oxidative phosphorylation pathway and reactive oxygen species (ROS) production in the relatively resistant CBA/Ca mice. We hypothesise that the increased ROS levels associated with higher levels of mitochondrial activity results in a highly oxidative cellular environment that has a dramatic effect on the nematode’s ability to successfully sustain a parasitic association with its resistant host. Under infection, both strains had increased abundances in proteins associated with the oxidative phosphorylation pathway, as well as the tricarboxylic acid cycle, with respect to their controls, indicating a general stress response to Ascaris infection. Despite the early stage of infection, some immune-associated proteins were identified to be differentially abundant, providing a novel insight into the host response to Ascaris. In general, the susceptible C57BL/6J mice displayed higher abundances in immune-associated proteins, most likely signifying a more active nematode cohort with respect to their CBA/Ca counterparts. The complement component C8a and S100 proteins, S100a8 and S100a9, were highly differentially abundant in both infected strains, signifying a potential innate immune response and the importance of the complement pathway in defence against macroparasite infection. In addition, the signatures of an early adaptive immune response were observed through the presence of proteins, such as plastin-2 and dipeptidyl peptidase 1. A marked decrease in proteins associated with translation was also observed in both C57BL/6J and CBA/Ca mice under infection, indicative of either a general response to Ascaris or a modulatory effect by the nematode itself. Our research provides novel insights into the in vivo host-Ascaris relationship on the molecular level and provides new research perspectives in the development of Ascaris control and treatment strategies.     

Investigation of protein induction in tumour vascular targeted strategies by MALDI MSI

Characterising the protein signatures in tumours following vascular-targeted therapy will help determine both treatment response and resistance mechanisms. Here, mass spectrometry imaging and MS/MS with and without ion mobility separation have been used for this purpose in a mouse fibrosarcoma model following treatment with the tubulin-binding tumour vascular disrupting agent, combretastatin A-4-phosphate (CA-4-P). Characterisation of peptides after in situ tissue tryptic digestion was carried out using Matrix-Assisted Laser Desorption/Ionisation-Mass Spectrometry (MALDI-MS) and Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Mass Spectrometry Imaging (MALDI IMS-MSI) to observe the spatial distribution of peptides. Matrix-Assisted Laser Desorption/Ionisation-Ion Mobility Separation-Tandem Mass Spectrometry (MALDI-IMS-MS/MS) of peaks was performed to elucidate any pharmacological responses and potential biomarkers. By taking tumour samples at a number of time points after treatment gross changes in the tissue were indicated by changes in the signal levels of certain peptides. These were identified as arising from haemoglobin and indicated the disruption of the tumour vasculature. It was hoped that the use of PCA-DA would reveal more subtle changes taking place in the tumour samples however these are masked by the dominance of the changes in the haemoglobin signals.     

Nonneutral evolution of tandem repeats in the mitochondrial DNA control region of lagomorphs

The mitochondrial DNA of the European rabbit (Oryctolagus cuniculus) contains a tandem array of 153-bp repeats in the vicinity of the replication origin of the H-stand. Variation among molecules in the number of these repeats results in inter- and intraindividual length polymorphism (heteroplasmy). Generally, in an individual, one predominant molecular type is observed, the others representing a low percentage of the mtDNA content. At the tissue level, we observe a particular distribution of this polymorphism in the gonads compared with liver, kidneys, or brain, implying a relationship between the differentiation status of the cells and the types of new mtDNA molecules which appear and accumulate during lifetime. Similar tandem repeats were also found in the mtDNA noncoding region of European hares (Lepus europaeus), a cottontail (Sylvilagus floridanus), and a pika (Ochotona rufescens). The lengths and the sequences of these units evolve rapidly and in a concerted way, but the number of repeats is maintained in a narrow range, and an internal 20-bp segment is highly conserved. Constraints restrict the evolution of the primary sequence of these repeated units, the number of which is probably controlled by a stabilizing selection.      

Accuracy of diagnostic registers and management of chronic obstructive pulmonary disease: the Devon primary care audit

Background

Guidelines on COPD diagnosis and management encourage primary care physicians to detect the disease at an early stage and to treat patients according to their condition and needs. Problems in guideline implementation include difficulties in diagnosis, using spirometry and the disputed role of reversibility testing. These lead to inaccurate diagnostic registers and inadequacy of administered treatments. This study represents an audit of COPD diagnosis and management in primary care practices in Devon.

Methods

Six hundred and thirty two patients on COPD registers in primary care practices were seen by a visiting Respiratory Specialist Nurse. Diagnoses were made according to the NICE guidelines. Reversibility testing was carried out either routinely or based on clinical indication in two sub-samples. Dyspnoea was assessed. Data were entered into a novel IT-based software which computed guideline-based treatment recommendations. Current and recommended treatments were compared.

Results

Five hundred and eighty patients had spirometry. Diagnoses of COPD were confirmed in 422 patients (73%). Thirty nine patients were identified as asthma only, 94 had normal spirometry, 23 were restrictive and 2 had a cardiac disorder. Reversibility testing changed diagnosis of 11% of patients with airflow obstruction, and severity grading in 18%. Three quarters of patients with COPD had been offered practical help with smoking cessation. Short and long-acting anticholinergics and long acting beta-2 agonists had been under-prescribed; in 15–18% of patients they were indicated but not received. Inhaled steroids had been over-prescribed (recommended in 17%; taken by 60%), whereas only 4% of patients with a chronic productive cough were receiving mucolytics. Pulmonary rehabilitation was not available in some areas and was under-used in other areas.

Conclusion

Diagnostic registers of COPD in primary care contain mistakes leading to inaccurate prevalence estimates and inappropriate treatment decisions. Use of pre-bronchodilator readings for diagnosis overestimates the prevalence and severity in a significant minority, thus post bronchodilator readings should be used. Management of stable COPD does often not correspond to guidelines. The IT system used in this study has the potential to improve diagnosis and management of COPD in primary care.